ABSTRACT Survivors of first primary breast cancer (BC) have a 2- to 5-fold increased risk of developing a second primary in the opposite breast. The high incidence and improved survival for BC has resulted in increasing numbers (currently nearly 3 million in U.S.) of women at risk of contralateral breast cancer (CBC). Studies to date have identified environmental, genetic, and treatment-related risk factors for CBC. However, despite advances in characterizing the etiology of CBC, a large fraction of CBC incidence remains unexplained, hindering the development of effective risk stratification or risk prediction tools which will be pivotal in developing effective CBC prevention, surveillance, and therapeutic strategies for BC survivors. In the context of CBC risk, first primary tumors reflect the joint effects of environmental exposures and host features, including those that are known and measurable as well as those that are unidentified and/or unmeasurable. Molecular features of first primary tumors could be powerful complementary indicators of risk of CBC. In a strategy designed to advance risk stratification capacity, we will use cutting edge genomic technologies to interrogate first primary tumors in the context of CBC risk. We will also assess the concordance of molecular features in paired first and second primary tumors to elucidate insights into the biology of CBC and potentially shared etiologic mechanisms. The proposed research builds on the unique resources of the large multi-center WECARE Study of CBC with features including: population-based ascertainment of CBC cases and controls (women with unilateral breast cancer (UBC)); detailed clinical data including treatment of first primary BCs from medical record reviews; extensive risk factor data; germline genetic data from an ongoing GWAS; and mammographic density data. We will obtain tumor blocks for first primary breast tumors of >500 UBC controls and first primary tumors plus contralateral tumors from > 500 CBC cases. Laboratory work will include: pathology reviews; transcriptome-wide molecular profiling of tumors; IHC analyses; and replication analyses. Our Primary Aim is to identify molecular biomarkers in first primary breast tumors associated with risk of developing a subsequent CBC. We will conduct: RNA-sequencing on first primary tumors; identify the most informative markers; develop a molecular signature associated with CBC and evaluate it jointly with treatment and lifestyle/personal/medical/germline genetic risk factors; and replicate with similar risk factors in an independent sample of > 400 CBC cases and > 400 UBC controls. Our Secondary Aims are to: (1) examine the risk signature in first primary tumors (established in the Primary Aim) in relation to CBC subtype-specific risk; and (2) assess the concordance of gene/transcript expression or alterations and subtypes in paired first primary and contralateral tumors and determine the extent to which lifestyle/personal/medical/germline genetic risk factors and treatment affect the development of marker-concordant CBC.